(1) Field of the Invention
The present invention relates to a method for crystallizing insulin or insulin analogs under alkaline conditions in the presence of zinc, purifying the crystals by filtering through a filter, and drying the crystals captured on the filter to produce crystalline insulin or insulin analog compositions.
(2) Description of Related Art
Diabetes mellitus is a chronic metabolic disorder caused by either a deficiency of insulin generated by pancreatic beta cells (Type 1) or an acquired cellular resistance to insulin (Type 2). Both Type 1 and Type 2 diabetes result in hyperglycemia, which can in turn result in long term complications. Since the introduction of insulin in 1921, all forms of diabetes have become treatable.
It is well known in the art that insulin may be crystallized in the presence of zinc ions, resulting in a crystalline preparation with significant benefits over amorphous, uncrystallized insulin with regards to stability, storage, formulation, and/or administration. Methods for crystallizing insulin lispro have been disclosed in U.S. Pat. Nos. 5,952,297; 5,028,587; 5,504,188; 5,597,893; 5,952,297 and 7,193,035. Methods for crystallizing an insulin analog in the absence of zinc ions is disclosed in U.S. Pat. No. 7,193,035, which discloses zinc-free crystallization of an insulin analog performed at a pH in the range of about 4.0 to about 7.5. Methods for crystallizing mixed crystals of insulin and insulin derivatives is disclosed in U.S. Pat. No. 5,028,587.
In the presence of zinc, human insulin self-assembles into stable hexameric structures (Chawdhury et al., Diabetologia 1983, 25, 460-464; Smith et al., Proceedings of the National Academy of Sciences 1984, 81, 7093-7097). Upon injection, the dissociation of human insulin hexamers into dimers and monomers is the rate-limiting step in absorption, causing a 30-45 minute delay in onset of action after administration (Kang et al., Diabetes Care 1991, 14, 942-948). In insulin lispro, the reversal of lysine and proline at positions 28 and 29 of the B-chain destabilize insulin dimer interactions (Ciszak et al., Structure 1995, 3, 615-622). When stabilized by a phenolic additive, hexameric insulin lispro preparations exhibit comparable stability to human insulin (Derewenda et al., Nature 1989, 338, 594-596; Bakaysa, D. L.; Radziuk, J.; Havel, H. A.; Brader et al., Protein Science 1996, 5, 2521-2531). However, after injection and dissipation of the phenolic preservative, hexameric insulin lispro dissociates into monomers more quickly than human insulin, resulting in a more rapid onset of action (Birnbaum et al., Pharmaceutical Research 1997, 14, 25-36).
Zinc content plays an important role in chemical and physical stability of pharmaceutical insulin formulations (Brange & Langkjoer, In Stability and characterization of protein and peptide drugs: Case histories, Wang, Y. J.; Pearlman, R., Eds. 1993; pp 315-350). Relative to monomeric insulin, hexameric preparations have greatly reduced susceptibility to chemical degradation, including deamidation (Brange et al., Pharmaceutical Research 1992, 9, 715-726) and covalent aggregation (Brange et al., Acta Pharmaceutica Nordica 1992, 4, 149-158). Physical stabilization of the hexamer structure by zinc also reduces the propensity for fibrillation (Brange et al., Diabetic Medicine 1986, 3, 532-536), while excess zinc causes insulin to precipitate into a crystalline suspension (Hallas-Moller et al., Science 1952, 116, 394-398). Zinc content therefore impacts the shelf life, biological activity, immunogenicity potential, and physical form of insulin formulations (Brange & Langkjoer, Op. cit.). For pharmaceutical insulin lispro solutions, a precise ratio of 3 zinc atoms per insulin hexamer was determined to be optimal (U.S. Pat. No. 5,474,978).
Commercial insulin manufacturing processes typically include a crystallization step to convert purified insulin into solid form, providing increased stability for bulk storage prior to formulation and filling (Walsh, Applied Microbiology and Biotechnology 2005, 67, 151-159). Classical insulin crystallization processes involve preparation of an acidic solution containing organic acid (acetic or citric), approximately 2 g/L insulin, and zinc. The solution pH is then adjusted to near the isoelectric point of insulin (pH 5.5-6.0), which initiates crystal formation (U.S. Pat. No. 2,920,014) During development of insulin lispro, it was noted that these conditions did not result in crystal formation, likely due to the reduced propensity of insulin lispro to self-associate (Brems et al., Protein Engineering Design & Selection 1992, 5, 519-525.). When this process was modified to include a phenolic stabilizer (phenol or resorcinol, but not m-cresol) at approximately 0.2%, insulin lispro crystals were obtained (U.S. Pat. No. 5,504,188,). A zinc-free insulin lispro crystallization process was also developed based on the classical “pH 8.2 process”, in which crystalline insulin salts are obtained from insulin solutions containing alkali metal cations or ammonium cations at alkaline pH (optimally pH 8.2) (U.S. Pat. No. 3,719,655). To adapt the process for successful crystallization of insulin lispro, a phenolic stabilizer was added and the optimal pH adjusted to 9.0 (U.S. Pat. No. 5,597,893). Though successful, these crystallization processes featured somewhat restrictive design spaces and were not robust with respect to the choice of phenolic stabilizer used.
While there are methods available for crystallizing insulin and insulin analogs, there is a need in the art for alternative methods of crystallizing insulin and insulin analogs such as insulin lispro.